A holder for a nail polish bottle that allows the bottle to be tipped at any angle wanted by the user and that allows viewing of an applicator extracting polish from the bottle. It has a fixed post attached to a base that stabilizes the holder against unwanted motion during use. There is a pivoting post also. The top of the fixed post and the bottom of the pivoting post form a ball and socket joint that allows the pivoting post to tilt at any angle wanted by a user. The pivoting post is attached to a spring-loaded clip. The clip contains arms with non-slip inner surfaces. The arms have an arcuate hollow elliptical oval shape that allows the user to view the transferring of polish to the applicator so that a proper amount is transferred. The clip is opened by pressing its lever portions together to stress the spring. When the pressure on the lever portions ceases, the arcuate arms close to hold the bottle securely.

The field of the present invention relates to an improved nail polish bottle holder that can hold nail polish bottles of various sizes and shapes at any dispensing angle desired and does not obscure the view of the bottles contents. It is useful for dispensing nail polish in the nail polishing process.
Nail polish is a fast drying lacquer that is ordinarily applied to the nails from a small bottle using a brush applicator affixed to the closure cap of the bottle. Because of the fast drying nature the user is often in a hurry. Further, the user is confronted with the problem of drawing a small quantity of the lacquer repeatedly. The bottle size is an annoying factor further complicated by the users need to secure bottle with one hand, as the applicator is dipped to draw the desired fill of polish. The bottle is then set aside as the applicator applies its fill of polish to a fingernail. These steps are repeated until all the nails are polished.
It is obvious that this process is tedious, awkward, and frustrating, frequently resulting in unsatisfactory results from spills of the nail polish and smudges in the newly painted nail that occur as a result of the constant manipulation of the nail polish bottle. Applying the nail polish without having to hold the bottle is therefore desirable.
It is a well-known phenomenon that when a brush is placed in a bottle of nail polish when the bottle is upright it is very difficult to extract the necessary amount of liquid from the bottle if the bottle is less then half full. However, if the bottle is tipped, it is much easier to extract the liquid. As the contents of the bottle reduce, the bottle must be tipped at slightly greater angles. A nail polish bottle holder that could hold the nail polish bottle at varying angles is therefore necessary.
Apparatus for holding the bottle at a desired angle allows the user more mobility to apply the polish resulting in a much better result. Since it is necessary to judge the depth of the inserted applicator in the nail polish bottle to avoid making a mess and drawing the required fill, it is desirable for the user to be able to see the contents of the bottle. Nail polish comes in bottles of various size and shape and therefore it is desirable to have a holder that can hold nail polish bottles of various size and shape.
These problems have drawn interest and patents have been granted on several inventions that address some of the problems of holding the bottle in a tipped position. However these inventions need further improvement. They tend to be limited to bottles of certain sizes or shapes or only allow the bottle to be tipped at certain angles. They may also call for the bottle to be held in an opaque frame that obscures sight of the liquid in the bottle, which makes it difficult to use an applicator properly. For example, LaBelle et al in U.S. Pat. No. 3,964,709 teach the use of a frame that keeps a bottle at generally 45xc2x0 from an upright position. DeBlasi et al in U.S. Pat. No. 5,141,188 teaches us the need to selectively tip the container of nail polish, however the invention is limited because the receptacle is lined with plastic foam and this will obscure the user""s view of the bottle interfering with the user""s ability to draw the desired amount of polish with the applicator. The device also is dependent on the use of detents to tip the bottle requiring more force that could result in smudges to the users newly painted nail. De Blasi also limits the size and shape of bottles that can be held, as does Siteman in U.S. Pat. No. 4,795,117, which is dependent on a sand bag of sufficient mass to restrain the bottle. Also, improper touch will upset the bottles tilt causing unsatisfactory results. The bottle support taught by Fleit, et al in U.S. Pat. No. 2,748,952 also limits the size of bottles. The present invention addresses the need for a nail polish bottle holder that does not obscure the view of the bottles contents and does away with the inability of prior art to hold nail polish bottles of varying size and shape at the desired angle.
The principal object of the present invention is to improve upon the prior art by providing a nail polish bottle holder that does not obscure the view of the bottles contents while holding bottles of various sizes and shapes at the angles desired to easily draw the nail polish from the bottle regardless of the amount of liquid remaining in the bottle. It is also the principal object of the present invention to eliminate difficulties in retrieving the nail polish from the bottle by eliminating the users need to hold the bottle while drawing the contents.
The present invention has four essential elements: a base, a fixed post; a pivoting post; and a clip. The fixed post has a bottom that is integrally attached to the base and has a top that forms a socket for a ball and socket joint. The clip is spring-loaded such that pressure must be applied to lever portions to open the arms of the clip, which have the shape of an arcuate hollow ellipse. If pressure is applied to the lever portions to open the arms, a nail polish bottle placed interior to the open arms, and then the pressure on the lever portions is released, the arcuate arms will press against the bottle and hold it securely. As the center of each arm is hollow the user can observe whether the brush applicator is extracting enough polish from the bottle, and therefore how much the bottle should be tilted. The pivoting post has a bottom section that is spherical in shape and that can be snapped into the socket in the fixed post to form a ball and socket joint. The rest of the pivoting post is generally cylindrical in shape with a lower section and an upper section. The upper section has a cross section that is smaller than the lower section except for lips at the top of the pivoting post. Also, there is a slit in the upper section. The clip is attached to the pivoting post by pushing the pivoting post up through the center of the clip""s flange and spring. The slit in the upper section is compressed to allow the top of the pivoting post to pass through the interior of the spring and the clip""s flange. After the slit has passed through the clip""s flange and spring, the clip is held securely on the pivoting post between the lips and the lower section. This operation and snapping the ball of the pivoting post into the socket of the fixed post, essentially assemble the bottle holder. A bottle held securely in the clip is tilted by tilting the pivoting post.
Another object of the present invention is to provide a device, which is of simple and inexpensive construction that can be easily assembled. Still another object of the present invention is to provide a nail polish bottle holder that allows easy insertion of nail polish bottles of various shapes and sizes. It is another object of the present invention is to provide a device that will eliminate unsatisfactory results. Another object of the present invention is to provide a device that allows the bottle to rotate on 360 degree around a center post and to tip 90 degrees from its vertical position. It is another object of the present invention to provide a device that is not archaic in appearance.


Durable non-electrically conductive metal treatments (such as coatings or finishes) for yarns and textile fabrics. Such treatments preferably comprise silver and/or silver ions; however, other metals, such as zinc, iron, copper, nickel, cobalt, aluminum, gold, manganese, magnesium, and the like, may also be present or alternatively utilized. Such a treatment provides, as one example, an antimicrobial fiber and/or textile fabric which remains on the surface and does not permit electrical conductivity over the surface. The treatment is extremely durable on such substrates; after a substantial number of standard launderings and dryings, the treatment does not wear away in any appreciable amount and thus the substrate retains its antimicrobial activity (or other property). The method of adherence to the target yarn and/or fabric may be performed any number of ways, most preferably through the utilization of a binder system or through a transfer method from a donor fabric to a target textile fabric in the presence of moisture and upon exposure to heat. The particular methods of adherence, as well as the treated textile fabrics and individual fibers are also encompassed within this invention.

This invention relates to improvements in durable non-conductive metal-based treatments (such as coatings or finishes) for yarns and textile fabrics. Such treatments preferably comprise silver and/or silver ions; however, other metals, such as zinc, iron, copper, nickel, cobalt, aluminum, gold, manganese, magnesium, and the like, may also be present or alternatively utilized. Such a treatment provides, as one example, an antimicrobial fiber and/or textile fabric which remains on the surface and does not permit electrical conductivity over the surface. The treatment is extremely durable on such substrates; after a substantial number of standard launderings and dryings, the treatment does not wear away in any appreciable amount and thus the substrate retains its antimicrobial activity (or other property). The method of adherence to the target yarn and/or fabric may be performed any number of ways, most preferably through the utilization of a binder system or through a transfer method from a donor fabric to a target textile fabric in the presence of moisture and upon exposure to heat. The particular methods of adherence, as well as the treated textile fabrics and individual fibers are also encompassed within this invention.
There has been a great deal of attention in recent years given to the hazards of bacterial contamination from potential everyday exposure. Noteworthy examples of such concern include the fatal consequences of food poisoning due to certain strains of Eschericia coli being found within undercooked beef in fast food restaurants; Salmonella contamination causing sicknesses from undercooked and unwashed poultry food products; and illnesses and skin infections attributed to Staphylococcus aureus, Klebsiella pneumoniae, yeast, and other unicellular organisms. With such an increased consumer interest in this area, manufacturers have begun introducing antimicrobial agents within various household products and articles. For instance, certain brands of polypropylene cutting boards, liquid soaps, etc., all contain antimicrobial compounds. The most popular antimicrobial for such articles is triclosan. Although the incorporation of such a compound within liquid or polymeric media has been relatively simple, other substrates, including the surfaces of textiles and fibers, have proven less accessible. There is a long-felt need to provide effective, durable, and long-lasting antimicrobial characteristics for textile surfaces, in particular on apparel fabrics, and on film surfaces. Such proposed applications have been extremely difficult to accomplish with triclosan, particularly when wash durability is a necessity (triclosan easily washes off any such surfaces). Furthermore, although triclosan has proven effective as an antimicrobial compound, the presence of chlorines and chlorides within such a compound causes skin irritation which makes the utilization of such with fibers, films, and textile fabrics for apparel uses highly undesirable. Furthermore, there are commercially available textile products comprising acrylic and/or acetate fibers co-extruded with triclosan (for example Celanese markets such acetate fabrics under the name Microsafe(trademark) and Acordis markets such acrylic fibers under the tradename Amicor(trademark)). However, such an application is limited to those types of fibers; it does not work specifically for and within polyester, polyamide, cotton, spandex, etc., fabrics. Furthermore, this co-extrusion procedure is very expensive.
Silver-containing inorganic microbiocides have recently been developed and utilized as antimicrobial agents on and within a plethora of different substrates and surfaces. In particular, such microbiocides have been adapted for incorporation within melt spun synthetic fibers, as taught within Japanese unexamined Patent Application No. H11-124729, in order to provide certain fabrics which selectively and inherently exhibit antimicrobial characteristics. Furthermore, attempts have been made to apply such specific microbiocides on the surfaces of fabrics and yarns with little success from a durability standpoint. A topical treatment with such compounds has never been successfully applied as a durable finish or coating on a fabric or yarn substrate. Although such silver-based agents provide excellent, durable, antimicrobial properties, to date such is the sole manner available within the prior art of providing a long-lasting, wash-resistant, silver-based antimicrobial textile. However, such melt spun fibers are expensive to make due to the large amount of silver-based compound required to provide sufficient antimicrobial activity in relation to the migratory characteristics of such a compound within the fiber itself to its surface. A topical coating is also desirable for textile and film applications, particularly after finishing of the target fabric or film. Such a topical procedure permits treatment of a fabric""s individual fibers prior to or after weaving, knitting, and the like, in order to provide greater versatility to the target yarn without altering its physical characteristics. Such a coating, however, must prove to be wash durable, particularly for apparel fabrics, in order to be functionally acceptable. Furthermore, in order to avoid certain problems, it is highly desirable for such a metallized treatment to be electrically non-conductive on the target fabric, yarn, and/or film surface. With the presence of metals and metal ions, such a wash durable, non-electrically conductive coating has not been available in the past. Such an improvement would thus provide an important advancement within the textile, yarn, and film art. Although antimicrobial activity is one desired characteristic of the inventive metal-treated fabric, yarn, or film, this is not a required property of the inventive article. Odor-reduction, heat retention, distinct colorations, reduced discolorations, improved yarn and/or fabric strength, resistance to sharp edges, etc., are all either individual or aggregate properties which may be accorded the user of such an inventive treated yarn, fabric, or film.
It is thus an object of the invention to provide a simple manner of effectively treating a yarn, textile, or film with a wash-durable antimicrobial metal or metal-ion containing treatment. A further object of the invention is to provide a treatment for textiles or films which is wash-durable and continuously reduces and/or removes malodors from the target surface through the utilization of metals or metal-ions. Another object of the invention is to provide an aesthetically pleasing metal- or metal-ion-treated textile or film which is non-electrically conductive, wash durable, non-yellowing, non-irritating to skin, and which provides either or both antimicrobial or odor-reducing properties.
Accordingly, this invention encompasses a treated substrate comprising a non-electrically conductive treatment comprising metal-containing compounds selected from the group consisting of metal particle-containing compounds, metal ion-containing compounds, and any combinations thereof, and a substrate selected from the group consisting of a yarn, a fabric comprised of individual yarns, and a film; wherein said compound or compounds is present on at least a portion of the surface of said substrate; and wherein at least about 30%, of the originally adhered metal-containing treatment remains on said treated portion of said substrate surface after at least 10 washes, said washes being performed in accordance with the wash procedure as part of AATCC Test Method 130-1981. Still more preferably at least 50% of the metal-containing compounds remain after 10 washes, more preferably 60% after 10 washes, and most preferably at least 75% after the same number of washes. Furthermore, it is also highly preferred that at least 30% of the finish is retained after 15 washes, 20 washes, and most preferably about 30 washes. Also, and alternatively, this invention encompasses a treated substrate comprising a non-electrically conductive treatment comprising metal-containing compounds selected from the group consisting of metal particle-containing compounds, metal ion-containing compounds, and any combinations thereof, and a substrate selected from the group consisting of a yarn, a fabric comprised of individual yarns, and a film; wherein said compound or compounds is adhered to at least a portion of the surface of said substrate; and wherein said treated substrate exhibits a log kill rate for Staphylococcus aureus of at least 1.5, preferably above 2.0, more preferably above 3.0, and a log kill rate for Klebsiella pneumoniae of at least 1.5, preferably above 2.0, and more preferably above 3.0, both as tested in accordance with AATCC Test Method 100-1993 for 24 hour exposure, after at least 10 washes, said washes performed in accordance with the wash procedure as part of AATCC Test Method 130-1981. Such an invention also encompasses the different methods of producing such a treated substrate. The wash durability test noted above is standard and, as will be well appreciated by one of ordinary skill in this art, is not intended to be a required or limitation within this invention. Such a test method merely provides a standard which, upon 10 washes in accordance with such, the inventive treated substrate will not lose an appreciable amount of its electrically non-conductive metal finish.
The amount retained may be measured in any standard manner, such as, for example, inductively coupled plasma (ICP), X-ray fluorescence (XRF), or atomic absorption (AA) spectroscopic analysis. Or, again, in the alternative, the durability of certain finishes may be determined (i.e., the retention of finish on the surface) in relation to antimicrobial performance. Thus, with an antimicrobially effective finish, the exhibition of log kill rates for Klebsiella pneumoniae or Staphylococcus aureus after 24 hours exposure in accordance with AATCC Test Method 100-1993 of at least 1.5, and higher, as noted above, for both after 10 washes in accordance with AATCC Test Method 103-1981. Preferably, these log kill rates are above 3.2, more preferably 3.5, and most preferably at least 4.0. Again, such log kill rates after the minimum number of washes symbolizes the desired durability level noted above.
Nowhere within the prior art has such a specific treated substrate or method of making thereof been disclosed, utilized, or fairly suggested. The closest art is a product marketed under the tradename X-STATIC(copyright) which is a fabric article electrolessly plated with a silver coating. Such a fabric is highly electrically conductive and is utilized for static charge dissipation. Also, the coating alternatively exists as a removable silver powder finish on a variety of surfaces. The aforementioned Japanese patent publication to Kuraray is limited to fibers within which a silver-based compound has been incorporated through melt spun fiber techniques. Nowhere has such a wash-durable topical treatment as now claimed been mentioned or alluded to.
Any yarn, fabric, or film may be utilized as the substrate within this application. Thus, natural (cotton, wool, and the like) or synthetic fibers (polyesters, polyamides, polyolefins, and the like) may constitute the target substrate, either by itself or in any combinations or mixtures of synthetics, naturals, or blends or both types. As for the synthetic types, for instance, and without intending any limitations therein, polyolefins, such as polyethylene, polypropylene, and polybutylene, halogenated polymers, such as polyvinyl chloride, polyesters, such as polyethylene terephthalate, polyester/polyethers, polyamides, such as nylon 6 and nylon 6,6, polyurethanes, as well as homopolymers, copolymers, or terpolymers in any combination of such monomers, and the like, may be utilized within this invention. Nylon-6, nylon-6,6, polypropylene, and polyethylene terephthalate (a polyester) are particularly preferred. Additionally, the target fabric may be coated with any number of different films, including those listed in greater detail below. Furthermore, the substrate may be dyed or colored to provide other aesthetic features for the end user with any type of colorant, such as, for example, poly(oxyalkylenated) colorants, as well as pigments, dyes, tints, and the like. Other additives may also be present on and/or within the target fabric or yarn, including antistatic agents, brightening compounds, nucleating agents, antioxidants, UV stabilizers, fillers, permanent press finishes, softeners, lubricants, curing accelerators, and the like. Particularly desired as optional and supplemental finishes to the inventive fabrics are soil release agents which improve the wettability and washability of the fabric. Preferred soil release agents include those which provide hydrophilicity to the surface of polyester. With such a modified surface, again, the fabric imparts improved comfort to a wearer by wicking moisture. The preferred soil release agents contemplated within this invention may be found in U.S. Pat. Nos. 3,377,249; 3,540,835; 3,563,795; 3,574,620; 3,598,641; 3,620,826; 3,632,420; 3,649,165; 3,650,801; 3,652,212; 3,660,010; 3,676,052; 3,690,942; 3,897,206; 3,981,807; 3,625,754; 4,014,857; 4,073,993; 4,090,844; 4,131,550; 4,164,392; 4,168,954; 4,207,071; 4,290,765; 4,068,035; 4,427,557; and 4,937,277. These patents are accordingly incorporated herein by reference. Additionally, other potential additives and/or finishes may include water repellent fluorocarbons and their derivatives, silicones, waxes, and other similar water-proofing materials.
The particular treatment must comprise at least one type of metal-incorporating compound (namely metal particles), metal-ion containing particles, or mixtures thereof. The term metal is intended to include any such historically understood member of the periodic chart (including transition metals, such as, without limitation, silver, zinc, copper, nickel, iron, magnesium, manganese, vanadium, gold, cobalt, platinum, and the like, as well as other types including, without limitation, aluminum, tin, calcium, magnesium, antimony, bismuth, and the like). More preferably, the metals utilized within this invention are generally those known as the transition metals. Of the transition metals, the more preferred metals are silver, zinc, gold, copper, nickel, manganese, and iron. Most preferred are silver and zinc. Such metals provide the best overall desired characteristics, such as, preferably, antimicrobial and/or odor reducing characteristics, certain colorations, good lightfastness, and, most importantly, wash durability on the target substrate.
The term metal particle is intended to encompass any compound within which the metal is present in its pure non-ionic state (thus silver particles are present, as one example). The term metal-ion containing encompasses compounds within which the ionic species of metals are present (such as metal oxides, including, as mere examples, zinc oxide for Zn2+, silver oxide for Ag+, and iron oxide for Fe2+ or Fe3+, or, as alternatives, ion-exchange resins, zeolites, or, possibly substituted glass compounds, which release the particular metal ion bonded thereto upon the presence of other anionic species). The preferred metal particle compound is produced through a reduction procedure and may be any of silver, nickel, copper, zinc, and iron. With a reducing agent, the salts utilized for this purpose are thus preferably silver (I) nitrate, nickel (II) perchlorate, copper (II) acetate, and iron (II) sulfate. The preferred metal-ion containing compound for this invention is an antimicrobial silver zirconium phosphate available from Milliken and Company, under the tradename ALPHASAN(copyright), although any silver-containing antimicrobial compound, including, for instance, and as merely some examples, a silver-substituted zeolite available from Sinanen under the tradename ZEOMIC(copyright) AJ, or a silver-substituted glass available from Ishizuka Glass under the tradename IONPURE(copyright), may be utilized either in addition to or as a substitute for the preferred species. Also preferred as such a compound is zinc oxide, zinc ricinoleate, zinc chloride, and zinc sulfate. Other metals, as noted above, may also be utilized; however, from a performance standpoint, silver and zinc, are most preferred. Generally, such a metal compound is added in an amount of from about 0.01 to 40% by total weight of the particular treatment composition; more preferably from about 0.05 to about 30%; and most preferably from about 0.1 to about 30%, all dependent upon the selected method of application. The metal compound is then added to the target substrate in a) amounts of between 0.01 and 1.0 ounces per square yard, or, alternatively, b) from about 0.01 to about 5% owf, depending on the selected application and method for measuring. Such proportions provide the best antimicrobial and/or odor-reducing performance in relation to wash durability, electrical non-conductivity, and overall cost. Preferably this metal compound add-on weight is a) about 0.1, or b) about 1.0% owf. The treatment itself, including any necessary binders, adherents, thickeners, and the like, is added to the substrate in an amount of a) about 0.01 to about 4.0 ounces per square yard, or b) from about 0.01 to about 10% owf.
Furthermore, the inventive substrates necessarily do not exhibit any appreciable electrical conductivity (due to the low amounts of metal present and thus the nonexistence of any percolation over or through the target substrate) as measured by attaching a two-inch by two-inch fabric specimen to two electrodes and applying a voltage gradient of about 100 volts per inch through the fabric (i.e., in accordance with AATCC Test Method 76-1978). The measured resistance in ohms per square inch should exceed about 10,000, preferably 1,000,000, and most preferably 1xc3x97109 in order to provide a substantially non-electrically conductive fabric.
The selected substrate may be any of an individual yarn, a fabric comprising individual fibers or yarns (though not necessarily previously coated yarns), or a film (either standing alone or as laminated to a fabric, as examples). The individual fibers or yarns may be of any typical source for utilization within fabrics, including natural fibers (cotton, wool, ramie, hemp, linen, and the like), synthetic fibers (polyolefins, polyesters, polyamides, polyaramids, acetates, rayon, acylics, and the like), and inorganic fibers (fiberglass, boron fibers, and the like). The target yarn may be of any denier, may be of multi- or mono-filament, may be false-twisted or twisted, or may incorproate multiple denier fibers or filaments into one single yarn through twisting, melting, and the like. The target fabrics may be produced of the same types of yarns discussed above, including any blends thereof. Such fabrics may be of any standard construction, including knit, woven, or non-woven forms. The films may be produced from any thermoplastic or thermoset polymer, including, but not limited to, polyolefins (polypropylene, polyethylene, polybutylene), polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, and the like, polyesters (polyethylene terephthalate, isophthalates, and the like) polyethers, acetates, acrylics, and polyamides, as well as any copolymer films of any of the above. Such films may be extruded, blown, rolled, and the like, and may be produced in situ on the surface of a target fabric or produced separately and subsequently adhered or laminated on a target surface. Also, such films may be produced, treated, and utilized separately from any other substrates.
The yarns are preferably incorporated within specific fabrics, although any other well known utilization of such yarns may be undertaken with the inventive articles (such as tufting for carpets). The inventive fabrics may also be utilized in any suitable application, including, without limitation, apparel, upholstery, bedding, wiping cloths, towels, gloves, rugs, floor mats, drapery, napery, bar runners, textile bags, awnings, vehicle covers, boat covers, tents, and the like. The inventive films may be present on fabrics, or utilized for packaging, as coatings for other types of substrates, and the like.
Yarn and fabric substrates are preferably treated with a metal particle or metal-ion containing finish. Films are preferably treated with metal-ion containing formulations on the surface of film-coated fabrics.
Metal Particle Treatments
The preferred metal particle composition will generally comprise four components: water, a metal salt, a reducing agent, and a polymeric binder. As noted above, the metal is produced through the reduction of the metal ion upon dissolution of the metal salt in solution. This specific process actually blends two different technologies, specifically the formation of colloidal particles by chemical reduction and steric stabilization of such particles by surfactant or polymer and the modification of a fiber (or textile) surface through the utilization of a polymeric binder. In the instance, the steric stabilizer and the fiber (or textile) binder are the same polymeric compound.
Such a metal particle dispersion is generally produced as follows: A solution of the polymeric binder and water is produced having a polymer concentration between 0.1% and 20% (w/w). The solution is then divided between two containers, one containing a dissolved metal salt (i.e. a metal salt MA dissociates completely to M+ and Axe2x88x92) and in the other, a dissolved reducing agent. When the two solutions are thoroughly mixed, they are then combined very quickly. When combined, the reducing agent transfers an electron to the metal cation and reduces it to its neutral form (Mn++exe2x88x92xe2x86x92Mn o). The metal atoms quickly agglomerate to form larger (1-1000 nm) particles. The steric stabilizer acts by adsorbing to the surface of the growing particles and thereby prevents catastrophic flocculation of the particles into macroscopic (xcx9cmm in diameter) aggregates by limiting the distance of closest approach.
It is important to note that the selection of the particular polymeric binder is crucial to the success in attaining the desired durability and effectiveness of the specific coating as this binder component must meet a number of important criteria. First, since high salt concentrations are necessary to generate large numbers of metal particles, and such salts generally cause many polymeric binder dispersions to flocculate out of solution, the particular binder must not react in such a manner in order to effectively stabilize the particles that are produced (as noted above). Secondly, the binder must not act like typical textile binders (which do not stabilize the particles and thus allow the nucleated particles to flocculate rapidly into macroscopic assemblies) which would render the resultant solution unusable in this application. Thirdly, it is important that the polymeric stabilizer, once processed, be able to withstand home washing under a wide range of conditions and maintain the silver concentration on the textile. Thus, it must not be readily soluble in water, must not be susceptible to attack by standard and/or industrial detergents, solvents, and/or bleaches, and must not melt upon exposure to drying temperatures. The utilization of such a specific binder to provide a metal coating to fibers and/or fabrics is thus drastically different from other previous practices in this area and permits a topical application of a such a coating either before or after the particular substrate has been finished. In order to provide the requisite wash durability, this binder must pass these stringent criteria. No teaching or fair suggestion exists within the prior art of such requirements.
As noted previously, the preferred metal salts for this procedure are silver (I) nitrate, nickel (II) perchlorate, copper (II) acetate, and iron (II) sulfate. The concentrations of these salts within the immersion bath can be increased to xcx9c2% before the kinetics of reduction and aggregation overwhelm the kinetics of polymer adsorption and mixing and cause significant aggregation/clumping of the metal. The preferred metal salt is silver (I) nitrate and is present in a concentration of from about 0.01% to about 10%, more preferably from about 0.1% to about 5.0%, and most preferably about 1.0% within the immersion bath.
The preferred reducing agents are sodium borohydride (NaBH4), sodium hydrosulfite, and trisodium citrate (Na3C6H5O7), although any standard reducing agent associated with the above-listed metal salts may be utilized. The former is a stronger reducing agent that reacts with the metal completely within seconds of mixing An undesirable byproduct of this reaction is hydrogen gas that causes significant foaming when mixed. The latter two do not have this effect, but are milder reducing agents and require heating to near boiling to cause the reaction to proceed.
The polymeric binder may be selected from certain resins and thermoplastics, such as melamine resins and polyvinyl chloride-containing polymers. Of particular interest, and thus the preferred polymeric binders for this process are melamine-formaldehyde resins (such as a resin available from BFGoodrich under the tradename Aerotex(copyright)), polyvinyl chloride/vinyl copolymers (such as a copolymer also available from BFGoodrich under the tradename Vycar(copyright) 460xc3x9749), and PVC/acrylic resins available from BFGoodrich. It has been found that upon exposure to an ammonium sulfate catalyst and curing at 350xc2x0 C. for 2 minutes, the melamine provides durable finish on either a fiber or a fabric of at least 30 washes. The copolymer requires no catalyst and performs similarly to the melamine in wash durability when cured for the same time and at the same temperature. (Table 1 lists the ICP reading for silver as a function of home washes using Aerotex(copyright) M3 and BFG Vycar(copyright) in a pad.)
The solution described above can be applied to fabric or yarn in a number of ways. Included in this list, which is by no means exhaustive, are pad coating, screen coating, spraying, and kiss-coating (particularly for yarn applications). The preferred coating and method are discussed in greater depth below.
Metal-Ion Containing Coatings
The preferred procedures utilizing metal-ion containing compounds include any of the following, depending on the desired characteristics of the final product. One alternative utilizes the silver-ion compound noted above, such as either ALPHASAN(copyright), ZEOMIC(copyright), or IONPURE(copyright) as preferred compounds (although any similar types of compounds which provide silver ions may also be utilized), exhausted on the target fabric or film surface and then overcoated with a binder resin. Alternatively, the metal-ion containing compound may be admixed with a binder within a dye bath, into which the target fabric or fiber is then immersed at elevated temperatures (i.e., above about 50xc2x0 C.).
Such a procedure was developed through an initial attempt at understanding the ability of such metal-ion containing compounds to attach to a fabric surface. Thus, a sample of ALPHASAN(copyright) was first exhausted from a dye bath on to a target polyester fabric surface. The treated fabric exhibited excellent log kill rate characteristics; however, upon washing in a standard laundry method (AATCC Test Method 130-1981, for instance), the antimicrobial activity was drastically reduced. Such promising initial results led to the inventive wash-durable antimicrobial treatment wherein the desired metal-ion containing compound would be admixed or overcoated with a binder resin on the target fabric surface. The binder resin should exhibit little or no water solubility (substantially water-insoluble), and must readily adhere to either the fabric surface or to the metal-ion containing compound itself. Such a binder resin can thus be selected from the group consisting of nonionic permanent press binders (i.e., cross-linked adhesion promotion compounds, including, without limitation, cross-linked imidazolidinones, available from Sequa under the tradename Permafresh(copyright)) or slightly anionic binders (including, without limitation, acrylics, such as Rhoplex(copyright) TR3082 from Rohm and Haas). Other nonionics and slightly anionics may be utilized as long as they provide the desired adhesion characteristics. Such potential compounds include melamine formaldehyde, melamine urea, ethoxylated polyesters, such as Lubril QCX(trademark) available from Rhodia, and the like. The initial exhaustion of ALPHASAN(copyright) is thus preferably followed by a thin coating of binder resin to provide the desired wash durability characteristics for the metal-based particle treatment. The antimicrobial characteristics of the treated fabric remained very effective for the fabric even after as many as ten standard laundering procedures. Also possible, though less effective as compared to the aforementioned binder resin overcoat, but still an acceptable method of providing a wash-durable antimicrobial metal-treated fabric surface, is the application of a metal-ion containing compound/binder resin from a dye bath mixture. The exhaustion of such a combination is less efficacious from an antimicrobial activity standpoint than the overcoat procedure, but, again, still provides a wash-durable treatment with acceptable antimicrobial benefits. In actuality, this mixture of compound/resin may be applied through spraying, dipping, padding, and the like.
Another preferred alternative concerns the treating of individual fibers. Such an alternative has proven very effective, most particularly in a package dyeing method. In such a procedure, a dye bath comprising the desired metal-ion containing compounds and binder agents is pumped through a tightly wound spool of yarn (or fiber). This is generally a rather difficult process to perform effectively since the particle sizes of the constituent dye bath solids might interfere with the requisite pump pressure to force the dye bath liquor through the entire xe2x80x9cpackagexe2x80x9d of yarn. Furthermore, such a dyeing method must produce a uniform treatment of all the portions of the target yarn throughout the xe2x80x9cpackagexe2x80x9d; particle size and thoroughness of mixing are thus of vital importance to impart of even treatment over the entire yarn. Surprisingly, this procedure works very well for imparting a wash-durable metal treatment on the yarn surface. Upon treatment of target yarns, such yarns can then be woven, knit, or incorporated within a non-woven fabric structure to form a textile. The textile exhibits similar colorations, log kill rates, and the like, at discrete locations of the textile. Without intending to be bound to any specific theory, it is believed that the binding agent appears to affix itself over the metal-ion containing compounds adhered to the yarn surface, or that the binding agent adheres to the yarn surface, to which the metal-ion containing compound then affixes itself (and to which binding agent may also affix). As such, and again, very surprisingly, the desired metal-ion containing compounds were small enough to be forced through the yarn xe2x80x9cpackagexe2x80x9d in order to treat the entire target yarn.
In such an alternative method, the high pressure procedure necessary for providing the antimicrobial solid application on the surface of the target yarns must be sufficient to permit penetration of the solid compounds into the actual yarn structure. A high temperature may be desired to permit xe2x80x9copeningxe2x80x9d of the fiber structure to facilitate such solids introduction within a solid yarn. In general, the high pressure conditions must be from about 0.1 and 100 pounds per square inch with an exposure time of from about 5 seconds to about 5 hours at a temperature in the range from about 25xc2x0 to about 325xc2x0 C. Such conditions are most readily provided within a jet dye, closed vessel system, and appears to work most readily for package dyed yarns. The type of fiber is consequential only to the extent that certain temperatures permit easier penetration within certain fibers. Thus, natural fibers (such as cotton) require relatively low temperatures to xe2x80x9copenxe2x80x9d of the cellulosic structure; nylon requires a much higher temperature (to exceed its glass transition temperature, typically) to provide the most effective antimicrobial characteristics. For the most part, the high pressure actually appears to force the solid particles into the yarns; surprisingly, such solid-solid interaction works to retain a substantial amount of the solid antimicrobial, even after washing. Preferably, however, a binder agent is added to aid in solid particle retention since such solid particles will most likely exhibit a desire to become detached from the yarn over time.
Another alternative utilizes a metal oxide treatment on a fabric or film surface, such as, preferably, zinc oxide, coated with a hydrophobic binding agent mixed with a synergistic amount of a polymeric hydrophilic material, applied to a fabric or film surface, primarily to provide a wash-durable odor-reducing finish on the target fabric. Generally, the presence of such a wash-durable hydrophobic binding agent is possible only on a hydrophobic fabric surface. However, hydrophobic fabric does not wick moisture and thus is very uncomfortable to wear (if utilized as apparel fabric) as compared to a hydrophilic moisture-wicking fabric. Also, such a hydrophobic binding agent overcoat for the metal oxide odor-reducing material would detrimentally mask the metal oxide surface and retard the odor neutralizing action of the effective compound, particularly when present on and/or in a wet fabric. However, in order to provide a wash-durable metal oxide odor-reducing finish to a textile or fabric, seemingly there is a need to provide a complete hydrophobic binding agent over the metal oxide. When such a xe2x80x9crequiredxe2x80x9d excess of hydrophobic agent binder is present, again, the metal oxide (i.e., zinc oxide) would most likely be encapsulated and thus will not contact the targeted odor-producing compounds. A reduction in such hydrophobic binding agent results in a lack of sufficient adhesion between the metal oxide particles and the target fabric to provide a wash-durable and wear-durable finish. It was found, surprisingly, that the necessary comfort (due to wicking of moisture) and simultaneous retention of sufficient adhesion are provided through the addition of a hydrophilic agent to the zinc oxide/binding agent formulation. With such an addition, a durable, moisture-wicking, and odor-reducing textile finish/coating is provided. Of great and surprising interest is the apparent synergistic interaction between the zinc oxide, binding agent and hydrophilic agent. The hydrophilic agent not only provides moisture-wicking comfort to the target fabric, but also facilitates the interaction of odor compounds in human perspiration with the preferred zinc oxide to allow efficient odor neutralization. In general the weight ratio of zinc oxide to resin binder is in the range of 100:1 to 1:100 to be effective. The ratio between 1:2 and 2:1 is preferred. One suitable wetting agent for this application may possess both hydrophilic and hydrophobic moieties within its structure. In such an instance, the specific hydrophilic moiety must be present in an amount sufficient to permit any moisture spread on the treated fabric surface. The finish also requires sufficient amounts of a hydrophobic moiety within its structure to make it water soluble. As a result, the finish will not be easily washed off in repeated washing cycles. Examples of such preferred wetting agents are sulfonated polyesters, ethylene oxide-propylene oxide copolymers, and ethoxylated polyesters. The preferred metal oxide is zinc oxide with fine particle size and high surface area. Fine particle size allows uniform distribution in an application medium and renders the treatment substantially transparent. Large particle zinc oxide tends to give a white background shade to a textile and therefore affects the appearance of the product. Particle sizes for this application should be preferably below 3 micrometers, most preferably, less than 1 micrometer. Zinc oxide high specific surface area is also preferred for this application. Preferred specific surface area of zinc oxide for this application is 10 m2/g or more.
The preferred embodiments of these alternatives fabric treatments are discussed in greater detail below.
Examples of particularly preferred compounds within the scope of the present invention are set forth below. None of the following inventive fabrics exhibited any electrical conductivity.
A) Silver-Particle Yarn and Fabric Treatments
The dispersions used in the durability and log kill study for the resultant articles with silver-particle treatments contained the following concentrations (all % are per weight of solution): 1% AgNO3, 0.5% NaBH4, 5% binder resin, 3% hydroxyethylcellulose thickener, and 90.5% water. The print pattern used was a 12 dpi dot pattern with each dot having xcx9c0.5 mm diameter circular shape.
Three sets of samples were tested at three different numbers of washes. The three sets were: a) untreated 100% polyester multifilament fabric, b) the same type of fabric treated with just the desired binder resin, and c) the same type of fabric treated with the above-described silver-particle dispersion. Each sample was tested for 0, 15 and 30 washes. To minimize the potential biocidal activity of the detergent, the 15 and 30 wash samples were run through two additional rinse cycles before analysis. In the pertinent Tables which follow, the log kill results were performed with a) an initial Staphylococcus aureus concentration of 3.8xc3x97106 CFU/mL and b) a Klebsiella pneumoniae inocculum initial concentration of about 18,000,000 CFU/mL.
The following three treatments were performed and shown to be create a highly washfast metal particle finish:
1) Pad Coating
The fabric article (100% polyester fabric) was dipped into a silver particle/polymeric dispersion comprising about 1 part of silver colloid solution and about 5 parts of a binder resin. Particular resins tested were Aerotex(copyright) M3, Vycar(copyright) 460xc3x9749, and a PVC/Acrylic resin (all resins available from BFGoodrich). The immersed fabric was then removed and run through a pad roll. The fabric was then heated to 350xc2x0 C. for 2 minutes. The resulting fabric was then first analyzed for particle count remaining on the fabric surface after treatment by ICP spectroscopy, both initially and after a number of washes utilizing the standard laundering procedure of AATCC Test Method 130-1981. The results are presented in tabular form below:
Thus, the retention of the metal finish was excellent for both binders (77% after 10 washes, 68% after 20, 55% after 30 for the PVC/Acrylic binder; 88% after 10 washes, 87% after 20, and 70% after 30). It should be noted that these measurements are subject to the variability within the measuring instrument as well; although they are considered relatively and should not deviate in any significant amount from the tabulated results, variations in results may occur. Furthermore, the treated fabrics were also tested for electrical conductivity through the method noted above (AATCC Test Method 76-1978); the noise of the measuring instrument exceeded any signal of the resistance measuring instrument, thus, the resistance is so high for the fabric that no appreciable conductivity was exhibited by all of the tested samples.
2) Yarn Application
In this method, the metal particle dispersion was applied utilizing a kiss-coater, which consists of a roll which constantly rotates in a bath of the metal dispersion. The roll transferred the solution to the top side of the roller, where an end of yarn passed against the roller and into an oven where it was cured at 350xc2x0 C. for 2 minutes then taken up onto a bobbin for further processing. The metal particle coated yarn was measured to be electrically non-conductive (by the spaced electrode method noted above) and typically included from about 20 to 30% by weight of the metal-particle dispersion. The silver-coated yarn was then knit or woven into a fabric with non-treated yarns at a ratio of 1 treated yarn to every 15 untreated yarns. The treated yarns were visible on only one side of the treated fabric and the resultant fabric exhibited excellent sustained antimicrobial performance. Table 3 shows ICP results for silver as a function of washes for a xe2x80x9csockxe2x80x9d knit from 70 denier treated yarn and 500 denier untreated yarn.
3) Screen Printing
In a screen printing application, the dispersion described above was thickened and pressed through a printing screen onto one side of a fabric in a finishing step. The preferred thickening agent for this Aqualon(copyright) Natrosol 99-250 HHR (in a concentration range of 1-10% by weight of solution) with the preferred concentration being 3% (which provides a desired intrinsic viscosity of from about 100,000 to about 400,000, preferably 200,000, centipoise at standard temperature and pressure). The viscosity of the metal particle/polymer dispersion may also be adjusted with the utilization of sufficient amounts of hydroxyethylcellulose; however, mixtures of HEC and the Aqualon(copyright) thickeners may prove sufficient to provide a resultant, preferred viscosity of 200,000 cps. Although preferred thickeners for screen printing have been found, one of ordinary skill in this art would appreciate that any number of acceptable thickeners may be utilized, either alone, or in combination, to provide the desired and/or necessity viscosity level in order to perform such a screen printing procedure. The thickened metal-particle containing dispersion was applied to the target fabric by squeezing it through a patterned rotary screen. The xe2x80x9ccoatedxe2x80x9d fabric was then cured at 350xc2x0 F. for at least 2 minutes to produce a coating that was washfast through at least 30 washes. Table 4 provides this durability data:
The treated fabric was then analyzed for its ability to provide antimicrobial effectiveness against Staphylococcus aureus and Klebsiella pneumoniae. The results were as follows:
The durable treatment not only retained its integrity over the target fabric surface, but also continued to provide an effective antimicrobial treatment as well.
B) Silver Ion Exchange. Silver Zeolite, and Zinc Oxide Fabric Treatments
As noted above, treatments of specific metal-ion containing compounds have proven to be wash-durable on certain yarn and fabric surfaces as well. These include the following, preferably with either the ALPHASAN(copyright) silver-based compounds or with zinc oxide. The following preferred embodiments exhibited resistivity measurements well in excess of 1xc3x97109 ohms per square inch of fabric in accordance with AATCC Test Method 76-1978.
1) Exhaustion of Compound Followed with Binder Resin Overcoat
a) Acrylic Binder Resinxe2x80x94A dispersion of ALPHASAN(copyright) (silver-based ion exchange compound available from Milliken and Company) was first produced through the mixing of about 30% by weight of the silver-based compound, about 23.0% by weight of a mixture of anionic surfactants, Tamol(copyright) SN, available from Rohm and Haas, and Synfac(copyright) 8337, available from Milliken and Company, and the remainder water. This dispersion was then applied through exhaustion within a dye bath to four fabric samples (all of 100% polyester construction; with 51 picks by 52 ends; 300 denier multifilament yarn). Two were dyed at a temperature of about 280xc2x0 F.; the others at a temperature of about 265xc2x0 F. The exhaustion level of the active ALPHASAN(copyright) compounds on the target fabrics was about 1.0% owf. The fabrics were then coated with an acrylic binder material, Rhoplex(copyright)TR3082, in an amount of about 2.5% owf. The coated fabrics were then heat-set at 380xc2x0 F. The log kill rate for unwashed fabrics for S. aureus was measured to be 4.9; for K. pneumoniae, 2.54. The results after multiple washings are tabulated below:
It is important to note, and as is well appreciated and understood by one of ordinary in the art, that variations in log kill rate measurements are prevalent, though, reliable, due to inherent difficulties in both biological testing and in the ability to establish completely controlled bacterium counts on such surfaces. These results thus show very favorable antimicrobial performance and thus excellent wash durability on the fabric surface.
b) Permanent Press Binder Resinxe2x80x94The same type of ALPHASAN(copyright) dispersion and exhaustion procedure was followed as above. The overcoat, however, was Permafresh(copyright), available from Sequa. Again, about 2.5% owf of this overcoat resin was applied over the ALPHASAN(copyright)-treated fabrics. Also added within the dye bath was a butyl benzoate carrier in an amount of about 2.5% owf. The log kill results for this sample were as follows:
Excellent durability results were thus obtained with such a system.
c) PD-92 Binder Resinxe2x80x94The same type of ALPHASAN(copyright) dispersion and exhaustion procedure was followed as above. The overcoat, however, was PD-92 available from Milliken and Company. Again, about 2.5% owf of this overcoat resin was applied over the ALPHASAN(copyright)-treated fabrics. Also added within the dye bath was a butyl benzoate carrier in an amount of about 2.5% owf. The log kill results for this sample were as follows:
Excellent durability results were thus obtained with such a system as well.
d) Effect of Increased amount of ALPHASAN(copyright) on Wash Durabilityxe2x80x94The same fabric treatments (with Permafresh(copyright) binder resin) as above were performed with the amount of ALPHASAN(copyright) increased to a 4% owf active addition to the target fabric surface (about 13.3% owf of the dispersion). The same padding on of the permanent press binder was followed as above. The log kill results for K. pneumoniae are as follows:
Again, excellent durability was obtained.
e) Effect of Increased amount of Permanent Press Binder Resin on Wash Durabilityxe2x80x94The same fabric treatments (with Permafresh(copyright) binder resin) as above were performed with the padded on amount of binder resin increased to a 7.5% owf addition to the target fabric surface. The log kill results for K. pneumoniae are as follows:
Again, excellent wash durability results were obtained.
2) Exhaustion of Compound with a Binder Resin
A dispersion of ALPHASAN(copyright) (silver-based ion exchange compound available from Milliken and Company) was first produced through the mixing of about 30% by weight of the silver-based compound, about 23.0% by weight of an anionic surfactant mixture of Tamol(copyright) and Synfac(copyright) 337 surfactant, and the remainder water. This dispersion was then applied through exhaustion within a dye bath which included an acrylic binder (Rhoplex(copyright) TR3082) which was present within the dye bath in a concentration of about 2.5% owf. A 100% polyester fabric (same as above) was then placed within the dye bath which was then heated to a temperature of about 280xc2x0 F. The exhaustion level of the active ALPHASAN(copyright) compounds on the target fabrics was about 1.0% owf. The fabrics were then heat-set at 380xc2x0 F. The log kill rate for unwashed fabrics for S. aureus was measured to be 2.35; for K. pneumoniae, 5.38. The results after multiple washings are tabulated below:
These results show very favorable antimicrobial performance and thus excellent wash durability on the fabric surface, though less favorable than for the resin overcoated fabrics.
3) Exhaustion of Other Silver-Based Compounds
The same general exhaustion methods were followed as above with the same padding on (denoted as P in the table below) and dye bath application (D in the following table) of a permanent press binder as above as well. The different silver-based compounds applied were AmpZ200 (a TiO2/silver metal product available from DuPont), and ZEOMIC(copyright) AJ80H. The add-on weights of these were the same 1.0% owf treatment as for the ALPHASAN(copyright) noted above. The durability results for these compounds were as follows for K. pneumoniae log kill rates:
These are excellent durability results, although not as good as for the ALPHASAN(copyright) treatments.
4) Package Dyeing Method
Again, as with al of the other inventive fabrics and yarns, the measured resistivity of the following yarns and fabrics exceeded 1xc3x97109 ohms per square inch.

Method of manufacturing yarns and fabrics having a wash-durable non-electrically conductive topically applied metal-based finish


A liquid cooled charging cable system may be provided. The liquid cooled charging cable system may comprise a source, a load, a liquid cooled charging cable, and a cooling device. The liquid cooled charging cable may connect the source to the load, and may supply electric energy from the source to the load. The liquid cooled charging cable may comprise a supply conductor and a return conductor. The cooling device may pump a coolant around the supply conductor and the return conductor where the supply conductor and the return conductor may be immersed in the coolant.

An electric vehicle (EV) charging system, also called an electric recharging point, a charging point, a charging station a charge point, and an EVSE (Electric Vehicle Supply Equipment), is an element in an infrastructure that supplies electric energy for recharging electric vehicles, such as plug-in electric vehicles, including electric cars, neighborhood electric vehicles, and plug-in hybrids. Because plug-in hybrid electric vehicles and battery electric vehicle ownership is expanding, there is a growing need for widely distributed publicly accessible charging stations, some of which support faster charging at higher voltages and currents than are available from residential EVSEs. Many charging stations are on-street facilities provided by electric utility companies or located at retail shopping centers and operated by private companies. These charging stations provide one or a range of heavy duty or special connectors that conform to the variety of electric charging connector standards.


Intracellular translocation of proteins, particularly protein kinase C (PKC) isoenzymes, provides a surrogate test system for determining toxicity of candidate compounds. The profile of translocation with respect to at least one and preferably two or more signal transduction proteins can be correlated with that of known toxins. In addition, databases of such profiles with respect to toxins of various types provide a useful set of standards for evaluating toxicity of candidate compounds. Moreover, to the extent that a toxin""s profile mimics that found in a diseased state, the toxin can be used to construct screens for compounds alleviating the disease.

The invention relates to methods to evaluate toxicity of pharmaceuticals or other compounds intended for human or animal use. In particular, it concerns evaluating the toxicity of candidate compounds by assessing the effect of these compounds on localization of signal transduction proteins, particularly the protein kinase C (PKC) isoenzymes. Such assays can then be used to identify antidotes. To the extent that a disease state can be mimicked by a toxin, the invention further provides an assay for drug discovery.
The value of recognizing the toxicity of compounds intended as pharmaceuticals, cosmetics, foods, or other applications where the compounds come into contact with humans or other animals is evident. Because of ethical considerations, not to mention economic ones, of using animal models to predict toxicity against humans, a number of surrogate toxicity tests have been developed permitting more efficient and less controversial approaches to evaluating the capacity of compounds to impact the viability or well being of organisms. For example, a human keratinocyte cell line (SVK-14) has been validated as a model for the effects of mustard vesicants on basal epidermal keratinocytes (Smith, C. N., et al., Human Exp Toxicol (1997) 16:247-253). Normal human epidermal keratinocytes in vitro have also been used to monitor sulfur mustard damage to nuclei and mitochondria (Cook, J. R., et al., Toxicol Pathol (1997) 25:481-486). Apoptosis and necrotic cell death have also been studied as a result of contact with toxic compounds (Kressel, M., et al., Cell Tissue Res (1994) 278:549-556).
The cosmetics industry has been particularly concerned with substituting in vitro tests for in vivo evaluations of toxins. LeClaire, J. et al. Toxicol Lett (1998) 102-103:575-579 describes the experience of L""Oreal in substituting an in vitro system whereby Langerhans cells are introduced into reconstructed epidermis as an alternative test for skin sensitization. The article states that L""Oreal was able totally to ban animal testing on cosmetic products. However, Benassi, L. et al. Contact Dermatitis (1999) 40:38-44 describes a similar attempt to provide a model for whole animal testing. The validity of a monolayer culture system of human keratinocytes was used as a surrogate for in vivo acute skin irritancy tests for several compounds. The results did not entirely correlate, and the authors conclude that this suggests that the keratinocyte monolayer cell culture technique cannot directly replace in vivo methods. Further, Curren, R. et al. in an abstract published in Environ Health Perspect (1998) 106 (Suppl. 2):419-425 reviews the current state of in vitro testing and concludes that in order to progress in the areas of eye and skin irritation, it is necessary to expand knowledge of toxic markers in humans and the biochemical basis of irritation. Thus, attempts to replace in vivo testing with in vitro models have not been entirely successful.
Other approaches will be familiar to the practitioner. Gene expression profiles obtained in response to treating cells or tissues with toxins or other compounds are commonly used as indices of the effects of these compounds. Gene expression can be evaluated either by assessing the pattern of distribution of various mRNAs or by looking directly at the protein levels or activities that result. U.S. Pat. No. 5,811,231 (Xenometrix) discloses a surrogate marker comprising detection of gene transcription from any of several disclosed xe2x80x9cstress responsexe2x80x9d promoters. It is desirable to create surrogates for other kinds of toxicity, such as hypertension, for example, which requires correlating molecular and physiological phenotypes. One such surrogate is disclosed in U.S. Pat. No. 5,569,588 (Acacia) which discloses methods for drug screening by providing a plurality of separately isolated cells, each having an expression system with a different transcriptional regulatory element. Contacting this plurality of cells with a drug candidate and detecting reporter gene product signals from each cell provides a profile of response to the drug with regard to this multiplicity of regulatory elements. In addition, U.S. Pat. No. 5,777,888 (Acacia) describes a system for generating and analyzing a stimulus response output from a collection of signals. The description includes artificial intelligence systems such as expert systems and neural networks for this purpose. These methods, however, rely on gene transcription which is a downstream effect from protein localization as provided hereinbelow.
Since mRNA abundance is now known to be poorly correlated with protein abundance in many instances (Gygi, S. P., et al. Mol Cell Biol (1999) 19:1720-1730 which is incorporated herein by reference), direct examination of the proteins is useful. Standard methods to analyze thousands of proteins in parallel, typically using 2-D gels and mass spectroscopy, are complicated by the frequent occurrence of post-translational modifications. Since the functional impact of many of these modifications is to change subcellular localization, direct visualization of protein location simplifies the analysis. Since degradation can be considered a translocation to the void compartment (typically, via the proteosome degradation machinery), abundance of the protein can be included in the same analysis. Thus, as used here, xe2x80x9cintracellular localizationxe2x80x9d includes a determination of protein levels and changes therein.
Structure activity relationships of compounds having similar toxicities have also been used to predict the behavior of compounds of analogous structures. In addition, cell-based high throughput assays wherein a flow cytometer can be used to identify dead cells, especially after staining with a membrane-impermeable dye, can be used to assess a response. None of these methods is based on assessing intracellular localization.
It has previously been suggested to use multivariate statistical methods to analyze toxicity data obtained from a multiplicity of assays. The National Cancer Institute has assayed over 60,000 compounds for cytotoxicity against 60 cell lines, thus creating an oncology oriented database. Compounds with similar mechanisms of cellular destruction show strong correlations in their activity profiles across the panel. For example, microtubule disrupting agents are particularly effective in some of the cell lines and particularly ineffective on others. A candidate compound which shows a similar pattern has a high probability of exerting an effect on microtubules. This panel was also evaluated with respect to specific responses to the compounds, such as the expression of particular proteins, for example, p53. A cluster of proteins was identified whose expression is correlated with poor efficacy of alkylating agents, but not correlated with efficacy of antitubulin drugs. Thus, by testing a candidate against the 60 cell lines and evaluating the expression of this cluster of proteins, it would be possible to predict whether the compound would or would not be an effective alkylating agent.
Many toxicities encountered clinically affect only a small fraction of the population, but the undetectable effects on the remainder of the population are undoubtedly more widespread. Therefore, assays which assess damage below the clinical threshold and below the threshold detectable by gene transcription are desirable.
There remains a need to develop systems that can serve as accurate surrogates for predicting a wider range of potential toxicity of candidate compounds, assessing the nature of the mechanism by which they exert their toxic effects, and evaluating efficacy of treatment protocols. The present invention offers such a system.
The present invention uses the intracellular distribution of proteins which are involved in signal transduction as a surrogate for evaluating toxicity. By focusing on intracellular localization in response to test compounds, the present invention offers the ability to provide a large number of data points, essentially simultaneously, so that a toxicity xe2x80x9cfootprintxe2x80x9d of a particular compound can be obtained. Similarly, the efficacy of antidotes to the toxin can be assessed by evaluating the changes in the footprint when a proposed therapeutic protocol is tested against the surrogate. It is also an aspect of the invention to compare the footprint associated with a defined toxin with the footprint associated with a disease state. Where similar footprints are found, the toxin-induced footprint can be used as a model for the disease state and therapeutic protocols for the disease state evaluated accordingly.
In a particularly preferred embbdiment, the invention also takes advantage of the crucial role played by protein kinase C (PKC) isozymes in intracellular signaling. Because intracellular signaling is closely associated with both positive and negative metabolic effects, monitoring PKC activity provides an ideal basis for generating a broadly useable database of toxicity profiles. It is known that the PKC isozymes react to stimuli by altering their subcellular localization. The xe2x80x9cbefore and afterxe2x80x9d locations are distinctive for each isozyme in each cell type. Thus, assessing the subcellular localization of the 11 PKC isozymes in response to contact by potentially toxic compounds serves to create a profile for the response to individual compounds. By correlating the profiles of a multiplicity of compounds with their toxic behavior, a database can be created so that testing an individual compound with respect to its effect on PKC isoenzyme intracellular localization can serve as a surrogate for evaluating not only whether or not it is toxic, but the nature of its toxicity.
As stated above, the footprint of the toxin even with respect to the limited population of PKC enzymes may correlate with the footprint associated with a disease. In this instance, therapeutic protocols for the disease, as well as possible antidotes for the toxin, can be evaluated by assessing their effects on the intracellular localization footprint.
Thus, in one aspect, the invention is directed to a method to evaluate the toxicity of a candidate compound, which method comprises observing the intracellular localization of at least one PKC isoenzyme or other signal transduction protein in the presence and the absence of the candidate compound; comparing the intracellular localization pattern in the presence of the candidate compound with the intracellular localization pattern of said signal transduction protein in the presence of a known toxin, whereby similarity of the pattern observed with respect to the candidate compound to that observed for said known toxin identifies said candidate compound as a toxic compound. It is also likely that the candidate compound has a similar mechanism of action when the similarity in pattern is observed using more than one signal transduction protein and/or more than one cell type, and/or by measuring translocation as a function of time to obtain the profiles or footprints.
In another aspect, the invention is directed to a method to obtain a database of signal transduction protein localization profiles in response to toxic compounds, and to the database thus obtained. Typically, an individual toxin will be evaluated with respect to its effect on at least one and preferably more than one protein. If several proteins are used in the evaluation, a profile for a single toxin can be obtained. Additional toxins will have similar or different profiles.
In another aspect, the invention is directed to a method to refine toxin footprints by expanding the protein panel under inspection, followed by identification of the principal factors most strongly correlated with a particular toxicity.
In still another aspect, the invention relates to a method to identify a treatment protocol for a disease condition which method comprises identifying a cellular function the inhibition of which would ameliorate said disease condition, identifying, by the method of the invention, a toxin which inhibits said cellular function, thus identifying said toxin as a medicament to ameliorate the condition.
In still another aspect, the invention is directed to a method to identify an antidote for a toxin by assessing the ability of a candidate antidote to reestablish the normal intracellular localization footprint that has been distorted by the toxin. In instances where a disease state is characterized by distortion of the normal footprint, therapeutic protocols for the disease state can similarly be evaluated.
The invention methods rely on the ability of currently available wide-field microscopic techniques to assess the intracellular location of proteins, which can be labeled for observation as described below. Typically, proteins that vary their intracellular location in response to stimuli are xe2x80x9csignal transduction proteins.xe2x80x9d Typical of such proteins are the protein kinase C (PKC) isoenzymes. However, other proteins involved in signal transduction are known and their roster continues to grow as the processes of the living cell continue to be elucidated.
This roster includes, for example, cell surface receptors which often aggregate in response to specific stimuli. It also includes adaptive proteins, for example proteins with an SH2 or SH3 domain. These typically translocate to activated receptors. Additional proteins are then recruited into such a complex, typically expressing an enzymatic function, for example, phospholipases or cyclic nucleotide synthases. Of course, transcription factors move from cytoplasm to the nucleus, for example, steroid receptors, STATS, NF-xcexaB, AP-1 (fos-jun).
In addition, some proteins change organelle localization on damage to the organelle, for example cytochrome C leaks out of damaged mitochondria. Still other proteins traffic subcellularly, for example the cystic fibrosis chloride channel. About 5% of this channel per hour cycles from the cell surface into coated pits and thence into the cell interior. The GLUT-4 glucose transporter moves to the cell surface in response to signals generated initially by insulin binding to the receptor. Finally, the cytoskeleton and associated proteins change in a variety of ways, including an overall vectorial bias in orientation and in the position of the microtubule organizing center.
Such changes are illustrated below with respect to T-lymphocytes.
The toxicity of a particular compound may be assessed by the translocation of only a single signal transduction protein. However, even more useful is the pattern of intracellular translocations of a multiplicity of proteins caused by stimulation by a toxin or other probe or by an abnormality in the condition of the cell. This pattern of intracellular localization can be considered a profile or a xe2x80x9cfootprintxe2x80x9d of the response to a stimulus, for example, a toxin, or as a result of a disease or other abnormal condition. Changes in this footprint in response to various additional stimuli, therefore, can be used to evaluate efficacy of treatments with regard to abnormal conditions or antidotes to toxins.
The known patterns of protein kinase C distribution can be used as an illustration. According to the method of the invention, the cellular response to a compound to be tested for toxicity is measured in terms of the localization of at least one PKC isoenzyme. There are 11 PKC isoenzymes known. It is known that these isoenzymes mediate different biological effects. For all isoenzymes, however, it is believed that the translocation of the PKC, often from the cell soluble fraction to the cell particulate fraction, is required for activity (Mochly-Rosen, D., Science (1995) 268:247-251). Indeed, isozyme-specific antibodies have been used to determine the subcellular localization and stimulation-induced translocation of several PKC enzymes in human breast cancer cell lines (Disatnik, M-H., et al., Cell Growth Diff (1994) 5:873-880; in cultured rat cardiac myocytes (Disatnik, M. H., et al., Ex Cell Res (1994) 210:287-297; and in human dermal microvascular endothelial cells (hDMEC) (Zhou, L. Y., et al., J Invest Dermatol (1995) 107:248-252). For example, in hDMEC, after stimulation with the PKC agonist phorbol myristic acetate (PMA), the seven isoenzymes studied showed distinctive before/after localizations, as shown in Table 1.
It is apparent from these results that a useful profile of the cells""response to an individual compound can be created by observation of the translocation of PKC isoenzymes wherein the translocation profile may be partial or complete. Since such profiles can be correlated with toxic effects measured independently, the profile becomes a surrogate for assessing not only the toxicity of the compound per se, but the type of toxic response it will exhibit.
As used herein, with respect to the use of PKC isoenzymes as the substrate surrogates, the term xe2x80x9cprofilexe2x80x9d or xe2x80x9cfootprintxe2x80x9d for a particular compound refers to the pattern of translocation observed for a multiplicity of PKC isoenzymes and/or cell types and/or by measuring translocation as a function of time. While profiles are especially useful in identifying particular types of toxic activity, a simple mimic of a particular translocation for a single PKC in a single cell type, if properly validated, can be used as an index for toxicity, in the sense of a surrogate system for toxic activity per se.
For example, phorbol myristic acetate (PMA) binds to the C1 region on the regulatory domain of PKC. Oancea, E. et al. J Cell Biol (1998) 140:485-498 describes assaying this response by employing a fusion of the C1 region with green fluorescent protein (GFP). In response to PMA, this fusion protein moves from a diffuse cytoplasmic localization to a membrane bound one. Thus, the C1-GFP fusion can act as a reporter for the endogenous analog of PMA, which is diacyl glycerol (DAG). Similarly, the regulatory subunit of the cAMP-dependent kinase, PKA, dissociates from the catalytic subunit when cAMP levels rise, followed by translocalization of the catalytic subunit. A GFP fusion to the catalytic subunit would thus provide a reporter for cAMP. More broadly, any translocating domain can be linked to an observable protein, such as GFP, and act as a reporter for the translocation inducing signal. The observable aspects may also be inherent.
Similarly, as described above, additional signal transduction proteins can be labeled and their intracellular locations evaluated. The profile or footprint with respect to these proteins is defined similarly.
Means for assessing the intracellular localization of the signal transduction proteins are available in the art. This is, of course, apparent from the studies cited above. A preferred method for observation uses a commercially available wide-field deconvolution microscope marketed as the Delta Vision Microscope from Applied Precision (Seattle, Wash.). This is an illustrative instrument of a class of xe2x80x9cwide-fieldxe2x80x9d microscopes which utilize silicon chip charge-coupled device (CCD) technologies to convert light directly into digital signals. An improved form of this commercially available example is described in U.S. Patent application Serial No. 60/102,434 filed Sep. 3, 1998 and incorporated herein by reference.
Intracellular localization is typically performed using antibodies to the relevant protein. Many such antibodies are commercially available (Research and Diagnostic Antibodies, Berkeley, Calif.). The antibodies are attached to individual labels so that they can be distinguished. A particularly preferred approach to individually labeling reagents, such as these antibodies, is described in U.S. Ser. No. 09/146,984 filed Sep. 3, 1998 and incorporated herein by reference. Briefly, particulate xe2x80x9cbeadsxe2x80x9d used to label specific reagents such as the antibodies are provided in a multiplicity of hues by varying the ratios and amounts of individual fluorescent dyes coupled to the beads. By varying these amounts, a large number of different xe2x80x9chuesxe2x80x9d can be created, thus distinguishing antibodies coupled to beads of different hues.
Other methods for measuring translocation are set forth in U.S. patent application Ser. No. 09/144,609 filed Aug. 31, 1998, incorporated herein by reference, and in the continuation-in-part application of U.S. patent application Ser. No. 09/146,984, also incorporated herein by reference.
Methods for immunostaining the intracellular components and fixing the immunostained cells have been described by Zhou, L. Y., et al., J Invest Dermatol (1995) 107:248-252, cited above. Briefly, the cells are fixed in cold acetone, blocked with 1% normal growth serum for 1 hour prior to overnight treatment with isoenzyme-specific anti-PKC antibody. After washing, the specimens are prepared for microscopy by mounting in 90% glycerol in PBS with 0.1% azide and 3% diazabicyclo[2.2.2]octane (an antiquenching compound). The time courses of responses ranging from minutes to hours are developed for each of the isoenzymes in response to the best compounds or the toxin standards. Relevant categories for intracellular location include nuclear, perinuclear, diffuse cytoplasmic, cytoplasmic fibril-associated, and membrane-associated.
In order to assess the toxicity of an individual compound, however, the profile of translocated signal transduction proteins obtained in response to that compound must be correlated with profiles obtained from compounds of known toxicity. There is, of course, a litany of compounds known to be toxic. Suitable toxic compounds useful to create the database with regard to skin irritancy include, for example, ifosfamide, a cancer therapeutic mustard; phorbol myristic acid (PMA), a known PKC agonist with skin irritant properties; urushiol, the irritant in poison ivy. For other kinds of toxicity, suitable reference compounds include the enterotoxins of Staphylococci; the exotoxins of the Streptococci, other bacterial toxins such as cholera toxin, anthrax toxin, and toxins derived from E. coli, Salmonella, Shigella and Campylobacter. The mechanisms of many of these toxins are known. For example, the enterotoxins and exotoxins of the Staphylococci and Streptococci respectively, provoke effects in the immune system mediated by cytokines secreted from stimulated T-cells; other toxins act by disrupting signal transduction components including ADP ribosylating proteins, adenyl and guanylate cyclases, calcium and protein kinases.
The effects of toxins in specific cell death assays has been cited above. In assessing the effect of mustards on nuclei, the membrane-permeable SYTO nuclear stain is used; effects on metabolism are determined by using the nontoxic dye for mitochondria, Alamar blue. In a useful assay for apoptosis, a fluorescent label is attached to 3xe2x80x2 ends of fragmented DNA. Fragmented DNA distinguishes apoptosis from necrosis. In the early stages of apoptosis, DNA breaks are diffusely distributed in the entire nucleus except for the nucleolus with crescent-like accumulations beyond the nuclear membrane. In more advanced states, the nucleus is transformed into many round bodies with intense labeling. In necrosis, however, no DNA fragmentation occurs at the outset of cell death, only appearing 24 hours after cell death, long after the point when cell membrane integrity is lost.
Thus, by using suitable prior art assays, the nature of the toxic effects of individual toxins used to create the database can be assessed.
When profiles for a number of toxins have been obtained so as to provide a database, established pattern recognition techniques can be employed to rank order the signal transduction protein distributions or the timing thereof as predictors of apoptosis or other mechanisms of toxicity. Application of such techniques is aided by the fact that the microscope images are collected in digital form. These multivariate techniques are described, for example, in Duleba, A. J., et al., Semin Reprod Endocrinol (1996) 14:139-153; Binqet, C., et al., Rev Epidemiol Santxc3xa9 Publique (1998) 46:329-336. Thus, the entire database can be stored and manipulated in computer-readable form. A typical correlation available by such manipulation of the database is, for example, a localization pattern that is a diagnostic marker for eventual apoptosis in basal epithelial cells. Such correlations are possible because different signal transduction proteins mediate different biological effects typically with a reinforcing effect. In this way, toxicity assays can be performed more quickly and easily. Antidote screening is further enabled by such an improvement in throughput.
A particularly useful aspect of multivariate analysis is xe2x80x9cfactor analysis,xe2x80x9d used to distinguish xe2x80x9cdriverxe2x80x9d phenotypes from xe2x80x9cpassengers.xe2x80x9d The ideal marker for a particular toxin is always associated with the toxin and never observed in its absence. Most observed changes are passengers, responding to a driver phenotype. Since regulatory networks typically use balanced, opposing activities, the driver phenotype might, for example, be a concurrent increase in A with a decrease in B. The ratio of A:B represents the driver phenotype. In n dimensions, the driver phenotype can be more complex.
The ability to correlate a profile with a particular cellular effect is valuable on a macroscopic scale as well, since tissue pathology is a consequence of cellular responses. For example, both chemical and heat-induced skin bums share tissue level features although they are produced at different times after exposure. These features include erythema followed by subcutaneous edema which develops into blisters involving superficial layers of the skin which heal by scab formation. These features have been traced to separation of the basal layer from the overlying dermis to form small vesicles, which is caused by basal cell death. In the case of chemical burns, the dying cells show the hallmarks of apoptosis, including condensation of nuclear chromatin and perinuclear vacuolation.
Thus, by using a toxin-induced profile or footprint as a model for a preapoptotic state of basal cells, the effect of various therapeutic agents on the late stage tissue level disease can be predicted. Successful therapeutic agents will restore the footprint to that of a normal cell, or to one more closely resembling it.
The localization of any signal transduction protein may be used as an appropriate data point. However, the ubiquitous role of PKC in cellular signaling processes allows a wide range of toxicities to be monitored using just these proteins. More refined analysis (fewer false positives and more informative classification) is enabled by examination of larger numbers of proteins. That is, the perfect driver phenotype will include contributions by many individual proteins. If a particular PKC isozyme""s contribution represents 30% of the statistical variance in that ideal driver phenotype, then just using PKC alone is suboptimal but still useful. Moreover, working forward instead of backward, PKC alone provides a benchmark for evaluation of other potential contributors to the driver phenotypexe2x80x94the new protein must increase the percent variance accounted for. With the multihue tag approach described above, many thousands of proteins can be monitored concurrently. Multivariate regression of translocation phenotypes versus toxicity will identify that cluster of coordinated responses that accounts for the largest percent of variance. This cluster, or more precisely the dominant contributors, are an approximation to the perfect driver phenotype. Novel compounds can then be classified by the degree of overlap of their effects with various such coordinated responses.
As a simplified illustration, a chart of localization patterns for two PKC isoenzymes might be shown as below, with crosses indicating the effect of five known skin irritants.
In this example, the combination of data from both proteins is a more reliable predictor than either alone. That is, the driver phenotype=40% protein 1 (Nxe2x86x92F)+60% Protein 2 (Cxe2x86x92M).
Clearly, the more data points that compose the profiles, the more valuable and predictive are the conclusions drawn from changes in the footprint. The possibilities are extremely large. Considering only the 11 PKC isoenzymes, each of which can be in one of five arbitrarily designated places before and after treatment with a stimulant (25 combinations) there are 2511 categories, for more than the number of known toxins. If additional signal transduction proteins are included, the numbers are much larger. Thus, multivariate statistics and systems such as neural nets and fuzzy logic become essential to deal with the volume of data in a search for approximates to the ideal driver.
It has become clear that nearly all diseases and chronic conditions involve damaged proteins. The damage may be directly inherited in the genome or later acquired in the genome by mutation or may be directly acquired at the protein level. Thus, toxins may be found at a protein level which alter the pattern of proteins to mimic or overcome the diseased state or condition. Generally, it is more facile and direct to regulate the level of a disease model by applying and withdrawing toxin than by turning genes on and off. In this way, subthreshold disease states can also be identified in a small population, allowing extrapolation to expected supra-threshold toxicity rates expected, assuming Gaussian distribution of modifier factors. Moreover, agents that shift the network from the disease pattern to the subthreshold pattern can be identified.
The invention method takes advantage of the homeostatic nature of the network system that controls cellular metabolism. Perturbation of the normal state by a toxin activates proteins initiating the maximally perturbed state, but also proteins which facilitate a decay back to the normal condition, for example, when toxin is withdrawn. Disease conditions which negatively affect one such set of proteins should be compensated by the identified agent.
Thus, in summary, the method of the invention provides a simple surrogate method to test compounds for toxic effect by simply observing the translocation or the translocation profile characteristic of toxicity in response to the compound. It further permits the nature of the toxic reaction to be assessed by comparing the profile to the database created from profiles of toxins whose mechanism of action is known. Finally, it permits identification of medicaments which are useful in effecting inhibition of target metabolic processes that may be associated with disease.

Protein localization assays for toxicity and antidotes thereto